Mapping sequence differences between thimet oligopeptidase and neurolysin implicates key residues in substrate recognition

Kallol Ray, Christina S. Hines, David W. Rodgers

Research output: Contribution to journalArticlepeer-review

29 Scopus citations


The highly homologous endopeptidases thimet oligopeptidase and neurolysin are both restricted to short peptide substrates and share many of the same cleavage sites on bioactive and synthetic peptides. They sometimes target different sites on the same peptide, however, and defining the determinants of differential recognition will help us to understand how both enzymes specifically target a wide variety of cleavage site sequences. We have mapped the positions of the 224 surface residues that differ in sequence between the two enzymes onto the surface of the neurolysin crystal structure. Although the deep active site channel accounts for about one quarter of the total surface area, only 11% of the residue differences map to this region. Four isolated sequence changes (R470/E469, R491/M490, N496/H495, and T499/R498; neurolysin residues given first) are well positioned to affect recognition of substrate peptides, and differences in cleavage site specificity can be largely rationalized on the basis of these changes. We also mapped the positions of three cysteine residues believed to be responsible for multimerization of thimet oligopeptidase, a process that inactivates the enzyme. These residues are clustered on the outside of one channel wall, where multimerization via disulfide formation is unlikely to block the substrate-binding site. Finally, we mapped the regulatory phosphorylation site in thimet oligopeptidase to a location on the outside of the molecule well away from the active site, which indicates this modification has an indirect effect on activity.

Original languageEnglish
Pages (from-to)2237-2246
Number of pages10
JournalProtein Science
Issue number9
StatePublished - Sep 2002


  • Model
  • Neurolysin
  • Specificity
  • Substrate
  • Thimet oligopeptidase

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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